Intracellular Ca signaling in heart is a highly integrated process defined by the concerted function of numerous channels, pumps, transporters and buffers. Although several pathologies are associated with defects in Ca2+ handling, the specific mechanisms that link the deficiency to organ-level cardiac dysfunction are generally not well defined. One example is T-wave alternans (TW-Alt). TW-Alt is observed as alternating beat- to-beat changes in the T-wave of the electrocardiogram (ECG) and constitutes an important arrhythmogenic mechanism that can lead to sudden cardiac death. Likelihood of TW-Alt increases with tachycardia and is thought to be associated with abnormalities in intracellular Ca2+ handling and/or cellular metabolism. Despite years of study and debate, the mechanistic links between TW-Alt, tachycardia, intracellular Ca2+ handling and cellular metabolism are still unclear. One obstacle has been the absence of an experimental model system where all the salient factors can be explored in an integrated context (i.e. the working heart). To address this obstacle, a novel method (pulsed local field fluorescence microscopy or PLFF) was developed. This method allows very localized, high-resolution measurements of surface membrane potential and intracellular Ca2+ handling in Langendorff-perfused beating hearts where organ-level parameters (like heart rate, ECG, ventricular pressure) can be manipulated and measured. Multiple parameter real-time recording, simultaneous monitoring at multiple sites on the heart, and an integrative theoretical model are combined here to identify the molecular mechanisms that generate TW-Alt. Mouse heart is used in this proposal to more definitively establish mechanism using existing transgenic animal models. Preliminary results have led to the following hypothesis: In the mouse heart, insufficient Ca2+ uptake by the sarcoplasmic reticulum (SR) during tachycardia (or metabolic stress) produces intracellular Ca2+ release alternans (Ca-Alt). This first occurs in cells lining the endocardium and then progresses transmurally across the ventricular wall. This Ca-Alt generates beat-to-beat changes in Na- Ca exchanger activity producing beat-to-beat alterations in action potential (AP) repolarization. The resulting transmural difference in AP repolarization creates the TW-Alt observed in the ECG waveform.